XBB.1, BQ1.1 and atypical BA.4.6/XBB.1 recombinants predominate current SARS-CoV-2 Wavelets with flu-like symptoms in Cameroon: A snapshot from genomic surveillance

As of December 2022, Cameroon had observed a slight resurgence of COVID-19, raising concerns on genomic surveillance of related-SARS-CoV-2 variants under circulation. Following a laboratory-based survey, positive SARS-CoV-2 samples detected from December-2022 through March-2023 were processed for targeted sequencing at the Chantal BIYA International Reference Centre (CIRCB) in Yaoundé-Cameroon. From all positive cases detected, 13 were successfully sequenced (mean age 34 years, 70% female); the majority of the cases were unvaccinated (70%, 9/13) and symptomatic (92%, 12/13); all with flu-like symptoms (100%, 12/12). Following RT-PCR, the median cycle threshold was 22.23 [18–24] for the N gene; and 24.09 [20–26] for the ORF gene, underscoring high viral loads. Phylogenetic analysis of nucleotide sequences identified four major sub-variants in circulation, of which BA.5 (3/13), the recombinants BQ.1.1 (4/13), XBB.1 (4/13) and novel atypical variant of BA.4.6/XBB.1 (2/13). This snapshot surveillance indicates the introduction/emergence and circulation of new Omicron sub-variants, all accompanied by minor/mild symptoms. However, these new sub-variants and recombinants call for continuous genomic surveillance to prevent further resurgence of Covid-19 epidemiological wave.


Introduction
Since the start of COVID-19 pandemic, Cameroon has experienced up to 5 major waves [1][2][3]; with the first being reported from March to September 2020 and driven by the original Wuhan strain (100%); the second wave was reported from October 2020 to May 2021 and driven mainly by alpha and beta variants (90%); the third wave from June to October 2021 and driven mainly by delta variants (90%); the fourth wave from December 2021 to March 2022 and driven by the Omicron (B.1.1.529)variant (100%); while the fifth wave was reported from July 2022 to March 2023, with mild symptoms but without delineating the circulating viral strains [2,3].Of note, the Omicron (B.1.1.529)is a variant of SARS-CoV-2 reported to the World Health Organization (WHO) by the Network for Genomics Surveillance in South Africa on 24 November 2021 [4][5][6].It was first detected in Botswana and rapidly became the predominant variant in circulation worldwide, with different sub-variants that have emerged from the original omicron lineage [4,7,8].
Since December 2022, Cameroon has observed a slight resurgence of cases with flu-like symptoms, raising concern about the potential circulation of new SARS-CoV-2 variants of concern.It would therefore be of great relevance to ensure ongoing genomic surveillance to inform public health response on possible impact of circulating variants on disease severity and preventive measures at population level.In this present investigation, Cameroonian patients positive for COVID-19 with flu-like symptoms were enrolled within the frame of genomic surveillance supported by the EDCTP PERFECT-Study.Of note, the EDCTP-PERFECT study aimed at evaluating the diagnostic performance of SARS-CoV-2 assays on well-characterized COVID-19 cases, including variant detection (https://edctp-perfect-study.com/).

Study type and setting
A laboratory-based survey was conducted on COVID-19 PCR positive nasopharyngeal samples.Testing and sequencing were conducted at the virology Laboratory of the "Chantal BIYA" International Reference Centre in Yaounde ´, Cameroon.

Processing of the samples
RNA was extracted from 200 μL nasopharyngeal clinical swabs samples using the DaAn gene viral RNA Mini kit according to the manufacturer's protocol.SARS-CoV-2 positivity was confirmed by real-time PCR using the DaAn gene rRT-PCR assay.Targeted sequencing of the spike protein was then initiated on viral RNA extracts following genotyping specific for SARS-CoV-2, adapted from published in-house viral genotyping protocols.
the grant).The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.

Competing interests:
The authors have declared that no competing interests exist.The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the U.S. Centers for Disease Control and Prevention.The use of trade names and commercial sources is for identification only and does not imply endorsement by the U.S. Department of Health and Human Services.

Confirmation of SARS-CoV-2 positivity
RT-PCR was conducted using the DaAn gene assay as per manufacturer's instructions (Guangzhou, Guangdong Province, China).RT-PCR amplification was performed using QuantStudio qPCR Systems (Scientific Thermofisher).The protocol used probes targeting the open reading frame (ORF) gene and the nucleocapsid (N) protein gene, with a lower limit of detection of 500 copies/mL and an amplification reaction of 45 cycles.

Amplification and sequencing of SARS-CoV-2
The protocol for SARS-CoV-2 amplification and sequencing is detailed elsewhere [9].Briefly, viral RNA was reverse-transcribed and amplified using the kit One-Step Invitrogen (Super-Script One-Step for long templates RT-PCR; Foster City, CA) and 2 different primers (5'-3'); 38F (-GTC AGT GTG TTA ATC TTA CAA CCA G-) as the forward, and 1191R (-TGC ATA GAC ATT AGT AAA GCA GAG A-) as the reverse, (the given position refers to the Wuhan strain of SARS-CoV-2).For each PCR reaction, positive and negative controls were used to ensure the effectiveness of the reaction and the absence of cross-contamination, respectively.Amplification results were revealed after agarose-gel electrophoresis and positive results were kept for the sequencing process.PCR products were then purified through the ExoSAP-IT kit (Applied Biosystems, Lithuania).Sequencing was performed with four different overlapping primers: 38F (-GTC AGT GTG TTA ATC TTA CAA CCA G-), 514F (-TCT CAG CCT TTT CTT ATG GAC CT-), 655R (-CCT GAG GGA GAT CAC GCA CTA-) and 1191R (-TGC ATA GAC ATT AGT AAA GCA GAG A-).The sequencing product was purified by gel filtration chromatography using Sephadex G-50 resin (Sigma-Aldrich) to eliminate excess primers, unincorporated dideoxynucleotides (ddNTPs) and salts.Capillary electrophoresis was performed on Applied Biosystems 3500 genetic analyzer (Applied Biosystems, Tokyo, Japan).

SARS-CoV-2 sequence analysis
Sequences were aligned, assembled and edited by the reference sequence using Seqscape V2.7.Spike sequences were interpreted using the COV19 Stanford algorithm (https://covdb.stanford.edu).All the sequences obtain were blasted and analyzed on NCBI covid database (https://www.ncbi.nlm.nih.gov/activ) and subvariants were confirmed following molecular phylogeny by using MEGA 11 with reference sequences downloaded on the GISAID database (https://gisaid.org)through Audacity instant app.

Ethical considerations
The present research was approved by the Cameroon national committee for human health research (N˚2020/05/1227/CNERSH/SP); all participants gave their written consent at inclusion and the CIRCB General Directorate issued administrative authorization for the study.For purpose of confidentiality and privacy, data were collected and processed using unique identifiers, and secured in a password encrypted database, with restricted access only to key investigators.

Results
Our sample population was drawn from a total of 737 participants tested for SARS-CoV-2 real-time qPCR.Of these, 42 (5.7%)qPCR positive for SARS-CoV-2.Among the positive cases (n = 42), 13 samples were eligible for sequencing based on CT values (i.e. with a CT < 33 amplification cycles) (Fig 1).

Discussion
Our primary aim was to provide genomic signatures to substantiate the current epidemiological trend of SARS-CoV-2 infection in Cameroon, with evidence on variants that were All cases were screened between December 2022 and March 2023, which are in line with the global circulation of omicron sub-variants [5,7,8,10].This in turn justifies the low severity (mild symptoms), as the omicron wave was characterized by fewer patients admitted to hospital, less severe illness, and a lower case-fatality rate [4,5,7,8,[10][11][12][13][14].The mild symptoms observed despite higher viral loads and low vaccination coverage further support of the higher risk of viral transmissibility and low severity in a context of population immunity by viral exposure during previous waves [15].The mild symptoms, observed among immune-competent individuals, might be concerning among vulnerable populations [11][12][13][14].Interestingly, the most common mutations found in this study were G142D (100%), G339DH (100%) and the R346T (92.3%).Following interpretation from the Stanford COVID-19 database, G142D is a common N-terminal domain (NTD) mutation present in the delta and omicron variants that interferes with the neutralization of many NTD-binding monoclonal antibodies (mAbs); as for G339D, it is a receptor biding domain (RBD) core mutation that is present in the omicron variant whereas G339H is present in BA.2.75 and XBB which does not appear to reduce mAb susceptibility.Finally, R346T was reported from BA.4.6 lineage but not in the ancestral BA4/5 variant; it is associated with reduction in susceptibility to Cilgavimab/COV2-2130/ AZD1061 (a mAbs) in vitro database (https://covdb.stanford.edu).
From a molecular epidemiology perspective, sub-variants reported here were similar to the global trends.However, the two atypical recombinants (BA.4.6/XBB.1),confirmed by molecular phylogeny, indicate ongoing viral recombination (novel mutations) and risk of emergence of a new variant subsequently [6,7].Even though this study lays emphasis on few cases, our findings also call for further characterization of these potential new recombinants in order to fully describe their mutational patterns.These insights underscore the need to reinforce routine genomic surveillance to detect potential resurgence timely, the impact of novel mutations on vaccine/immune-escape, to mitigate global outbreaks.
The emergence and circulation of new Omicron sub-variants and recombinants are driving the current trend of COVID-19 epidemiology in Cameroon with mid symptoms.However, the emergence of atypical recombinants (BA.4.6/XBB.1)calls for routine genomic surveillance to timely detect and track novel strains, related disease severity and risk of transmission for optimal pandemic control.